DESCRIPTION: (Modified from the applicant's abstract). In salivary glands, chloride channels play a central role in fluid and electrolyte secretion by providing an apical Cl- efflux pathway. Three chloride channels have been characterized recently in rat parotid acinar cells, including an inward rectifying, hyperpolarization-activated channel that is thought to be involved in maintaining the resting membrane potential. The functional properties of this latter channel are similar to ClC-2, a member of the ClC gene family of voltage-activated chloride channels. Additional experiments showed that ClC-2 transcripts as well as ClC-2 proteins are expressed in rat parotid gland epithelia. A similar voltage dependence pattern for the parotid acinar cell channel and the expressed ClC-2 protein in HEK 293 cells suggests activation over the range of physiological membrane potentials. Consequently, activation of this channel during secretagogue-activated fluid secretion would tend to short-circuit the secretion process unless the channel is localized to the apical membrane. Thus, the author suggests that the ClC-2 channel must be tightly regulated during stimulated secretion in order to prevent short-circui of transepithelial Cl- movement. Abnormal regulation of this channel would result in salivary gland dysfunction, which will ultimately impact oral health To understand the regulatory mechanisms controlling the saliva secretion process, it is important to study the regulation of the ClC-2 chloride channel which is considered to be responsible for the hyperpolarization-activated chloride currents in rat parotid acinar cells. It has been recently reported that some ClC-2 homologues and ClC-2-like chloride currents in T84 cells are regulated by phosphorylation. The primary amino acid sequence of the cloned ra ClC-2 also contains several potential phosphorylation sites for different kinases. To determine whether CIC-2 proteins are substrates for kinases and to identify the functional sites in ClC-2, in vitro and in vivo labeling kinase assays of immunoprecipitated ClC-2 proteins and site-directed mutagenesis of the potential phosphorylation sites in ClC-2 will be performed. To investigate if phosphorylation of the ClC-2 protein modulates its activity, chloride currents from the wild type ClC-2 protein expressed in HEK 293 cells will be compared to those of expressed ClC-2 proteins containing mutations of the potential phosphorylation sites using patch clamping techniques. As proposed, the combined tools of molecular biological and electrophysiological techniques will allow the investigators to investigate the mechanisms regulating the activity of the ClC-2 protein and may aid in the development of treatments for patients suffering from dysfunction of fluid secreting epithelia including salivary glands.